Aryl hydrocarbon receptor (AHR) agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are some of the most toxic chemicals known to man. They also hold 4 of the top 10 positions within the EPA-ATSDR registry of priority substances that contaminate National Priority List. The toxicity of these compounds is primarily dependent upon the presence of a functional AHR signaling complex. This complex, in the absence of ligand consists of the AHR bound to a dimer of the heat shock protein of 90 kDa (Hsp90), the immunophilin-like protein, ARA9 (also known as XAP2 and AIP) and possibly several other factors (eg pp60src, p21). The role these chaperones play and their mechanism of action remains largely unknown. Our recent preliminary experiments suggest that ARA9 may function by recruiting other cellular factors to the AHR cytosolic complex. The role these cellular factors and other signaling systems play in the formation and integrity of the AHR cytosolic complex (upstream events) and how these other complex proteins influence AHR mediated toxicity (downstream events) has not been thoroughly explored. These signals may play important roles in the tissue specific biology and toxicity of AHR agonists. Our preliminary data and recent literature have led us to hypothesize: Secondary signaling, both upstream and downstream, plays an important role in AHR mediated signaling and toxicity through direct influence of the activity of the AHR cytosolic complex and perturbations of downstream signaling cascades. To address the hypothesis this project will look at the effects of secondary signaling on AHR biology in four specific aims (SA). SA1) Identify and characterize the proteins capable of interacting with the AHR in liver and immune cells in the absence and presence of ligand using tandem affinity purification, mass spectrometry and retroviral mediated gene transfer. SA2) Determine the fate of AHR complex members following ligand exposure using mass spectrometry and retroviral mediated gene transfer. SA3) Characterize the role of AHR-interacting proteins in ligand-induced signaling using RNAi, transient transfections and functional assays. SA4) Create a functional interaction network map for the AHR using proteins identified in the first aims and published reports and determine its overlap with regulatory networks. The completion of these aims will create a detailed picture of the AHR protein interaction network (AHR-PIN) and directly relate the proteins in this PIN to functional consequences for AHR mediated toxicity. Finally, the computational model that will be developed will generate new mechanistic directions for understanding the toxicity of AHR ligands and allow more accurate risk assessment for Superfund sites.